JP2019517787A - TrkB agonist antibodies for the treatment of neurodegenerative disorders - Google Patents

Abstract

The present invention provides TrkB agonist antibodies or antigen binding fragments that specifically enhance TrkB signaling activity. The present invention also promotes survival and regeneration of retinal ganglion cells, and treats subjects suffering from or at risk of developing various neurodegenerative coxiditions, such as glaucoma. Or provide therapeutic methods using such antibodies to prevent. [Selected figure] Figure 1

Description

Cross-Reference to Related Applications This patent application claims the benefit of priority from US Provisional Patent Application No. 62 / 331,046, filed May 3, 2016. The entire disclosure of this priority application is incorporated herein by reference in its entirety for all purposes.

  The Trk receptor family is important in physiological and disease processes in both neuronal and non-neuronal tissues. Among such receptor kinases, TrkB is a brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), which is essential for differentiation, survival and function of both peripheral and central nervous system neurons. And plays a pivotal role in neurotrophin-4 (NT4) signaling. Binding of the neurotrophin to the extracellular domain of the Trk receptor initiates a signal transduction pathway which results in dimerization and autophosphorylation of the receptor. Autophosphorylation of TrkB results in the activation of signaling pathways including mitogen-activated protein kinase (MAPK), phospholipase C-gamma and phosphatidylinositol-3 kinase (PI3-K).

  Both BDNF and TrkB have been shown to play a crucial role in the survival of retinal ganglion cells in the retina. It has been suggested that aberrant TrkB signaling and its suppression contribute to the development of various neurological and degenerative pathological conditions such as glaucoma. Glaucoma is the second most common eye disease, leading to blindness, affecting more than 60M people worldwide, and having a prevalence of approximately 2.3M diagnosed in the United States. Glaucoma results in damage to retinal ganglion cells (RGCs) and their axons (conveying all visual stimuli to the brain), mainly in "plumbing" problems leading to elevated intraocular pressure (IOP) . Although numerous drugs and devices for lowering IOP have been approved, they inhibit, retard or reverse cell atrophy and loss of RGCs or other retinal neurons and eventually cause blindness in> 10% of glaucoma patients Drugs that will be stopped are not yet developed. The ability to specifically modulate TrkB signaling may be crucial in various pathological scenarios associated with this pathway.

  There is a strong need in the art for means to promote Trk B and BDNF signaling activity in the treatment of various retinal diseases and disorders, particularly neurological disorders such as glaucoma. For example, current treatment options for glaucoma decrease IOP but not reverse RGC degeneration. The present invention is directed to addressing such and other needs.

  In one aspect, according to the present invention, a TrkB agonist antibody or antigen binding fragment that specifically binds to tropomyosin receptor kinase B (TrkB), wherein the TrkB agonist antibody or the same binding specificity as that of the second antibody or Providing an antigen binding fragment. The second antibodies are (1) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and SSRDSSRSHHLL (SEQ ID NO: 73). (2) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ARQGASSTSYDY (SEQ ID NO: 48), QSISSY (SEQ ID NO: 49), AAS and QQANSFPVA (SEQ ID NO: 50); (3) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56); ) GF (5) FSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKTDSDK (SEQ ID NO: 58) and AIWHSSAV (SEQ ID NO: 56); ) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 55), AIWHSSAV (SEQ ID NO: 56); 6) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSACV (SEQ ID NO: 59); (7) G IFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), ARQLLY (SEQ ID NO: 60), SGINVGAYR (SEQ ID NO: 57), YKSSDDK (SEQ ID NO: 61), AIWHSSAWV (SEQ ID NO: 56); ) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ATRVLPAGHF (SEQ ID NO: 62), NIGDKF (SEQ ID NO: 63), YDS and QVWDNSSNQGV (SEQ ID NO: 64); (9) VSDNSGAWN (SEQ ID NO: : 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and NSRDGSGNNVV (SEQ ID NO: 69); or (10) GYTFTSYA (SEQ ID NO: 70) Heavy chain CDRs 1 to 3 which are identical to INTNTGNP (SEQ ID NO: 71), ASRLAAAGFDY (SEQ ID NO: 72), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56) Contains the sequences of light chain CDRs 1-3.

  Some antibodies or antigen-binding fragments of the invention have heavy chain variable region sequences and light chain variable region sequences, one or both of which are respectively (1) SEQ ID NO: 38 and SEQ ID NO: 39; 2) SEQ ID NO: 14 and SEQ ID NO: 15; (3) SEQ ID NO: 16 and SEQ ID NO: 17; (4) SEQ ID NO: 18 and SEQ ID NO: 19; (5) SEQ ID NO: 20 and SEQ ID NO: 21 (6) SEQ ID NO: 22 and SEQ ID NO: 23; (7) SEQ ID NO: 24 and SEQ ID NO: 25; (8) SEQ ID NO: 26 and SEQ ID NO: 27; (9) SEQ ID NO: 28 and SEQ ID NO: (10) SEQ ID NO: 30 and SEQ ID NO: 31; (11) SEQ ID NO: 32 and SEQ ID NO: 33; (12) SEQ ID NO: 34 and SEQ ID NO: 35; or (13) SEQ ID NO: 36 Sequence of heavy chain variable region shown in SEQ ID NO: 37 and alignment of light chain variable region It is the same as. Some of the antibodies or antigen binding fragments are (1) SEQ ID NO: 38 and SEQ ID NO: 39; (2) SEQ ID NO: 14 and SEQ ID NO: 15; (3) SEQ ID NO: 16 and SEQ ID NO: 17; (4) SEQ ID NO: 18 and SEQ ID NO: 19; (5) SEQ ID NO: 20 and SEQ ID NO: 21; (6) SEQ ID NO: 22 and SEQ ID NO: 23; (7) SEQ ID NO: 24 and SEQ ID NO: 25 (8) SEQ ID NO: 26 and SEQ ID NO: 27; (9) SEQ ID NO: 28 and SEQ ID NO: 29; (10) SEQ ID NO: 30 and SEQ ID NO: 31; (11) SEQ ID NO: 32 to Sequence (12) SEQ ID NO: 34 and SEQ ID NO: 35; or (13) The heavy chain variable region sequences shown in SEQ ID NO: 36 and SEQ ID NO: 37 and the light chain variable region sequences.

  Some antibodies or antigen-binding fragments of the present invention are (1) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN, respectively. And SDRDSSRSHHLL (SEQ ID NO: 73); (2) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ARQGASSTSYDY (SEQ ID NO: 48), QSISSY (SEQ ID NO: 49), AAS and QQANSFPVA (SEQ ID NO: (3) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAWV (4) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKTDSDK (SEQ ID NO: 58) and AIWHSSAWV ( (5) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 55), AIWHSSAWV (SEQ ID NO: 56); (6) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSACV ( (7) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), ARQLLY (SEQ ID NO: 60), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 61), AIWHSSAWV (SEQ ID NO: 56); (8) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ATRVLPAGHF (SEQ ID NO: 62), NIGDKF (SEQ ID NO: 63), YDS and QVWDNSSNQGV (SEQ ID NO: 64) (9) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and NSRDGSGNNVV (SEQ ID NO: 69); or (10) ) GYTF Substantially the same as TSYA (SEQ ID NO: 70), INTNTGNP (SEQ ID NO: 71), ASRLAAAGFDY (SEQ ID NO: 72), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56). And heavy chain CDR1 to 3 and light chain CDR1 to 3 sequences that are identical. Some antibodies or antigen binding fragments have the sequences of heavy chain CDRs selected from the group consisting of SEQ ID NOs: 46-48, 51-53, 60, 62, 65-67 and 70-72. Some such molecules further comprise sequences of light chain CDRs selected from the group consisting of SEQ ID NOs: 49, 50, 54-59, 61, 63, 64, 68, 69 and 73. Some of such molecules are (1) SEQ ID NOS: 70-72, (2) SEQ ID NOS: 46-48, (3) SEQ ID NOS: 51-53, (4) SEQ ID NO: 51, 52, 60, (5) SEQ ID NOs: 46, 47 and 62, or (6) sequences of heavy chain CDRs 1-3 that are identical to SEQ ID NOs: 65-67. Some such antibodies or antigen binding fragments are each (1) SEQ ID NO: 65-68, GKN and SEQ ID NO: 73; (2) SEQ ID NO: 46-49, AAS and SEQ ID NO: 50; (3) SEQ ID NO: 51 to 56 (4) SEQ ID NO: 51 to 53, 57, 58 and 56; (5) SEQ ID NO: 51 to 53, 57, 55 and 56; (6) SEQ ID NO: 51 to 55 and 59; (7) SEQ ID NO: 51, 52, 60, 57, 61 and 56; (8) SEQ ID NO: 46, 47, 62, 63, YDS and SEQ ID NO: 64; (9) SEQ ID NO: 65 SEQ ID NOs: 70-72 and 54-56 have the sequences of heavy chain CDRs 1-3 and light chain CDRs 1-3 shown in SEQ ID NOs: 70-72 and 54-56, respectively.

  Some antibodies or antigen binding fragments of the invention are selected from the group consisting of SEQ ID NOs: 49, 50, 54-59, 61, 63, 64, 68, 69 and 73 and the sequences AAS, YDS and GKN. Contains the sequence of the chain CDRs. Some such molecules further comprise sequences of heavy chain CDRs selected from the group consisting of SEQ ID NOs: 46-48, 51-53, 60, 62, 65-67 and 70-72. Some such molecules are: (1) SEQ ID NO: 68, GKN and SEQ ID NO: 73; (2) SEQ ID NO: 49, AAS and SEQ ID NO: 50; (3) SEQ ID NO: 54 (4) SEQ ID NO: 57, 58 and 56; (5) SEQ ID NO: 57, 55 and 56; (6) SEQ ID NO: 54, 55 and 59; (7) SEQ ID NO: 57, 61 and 56; (8) SEQ ID NO: 63, YDS and SEQ ID NO: 64; or (8) SEQ ID NO: 68, GKN and the sequences of light chain CDRs 1 to 3 which are identical to SEQ ID NO: 69.

  Some antibodies or antigen binding fragments of the invention are heavy chains substantially identical to SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 Includes variable regions. Some antibodies or antigen binding fragments are scFv fragments, which contain a linker sequence linking heavy and light chain variable region sequences. Some exemplary linker sequences are shown in SEQ ID NOs: 40-45. Some scFv fragments of the invention comprise the sequence set forth in any one of SEQ ID NOS: 1-13.

  The antibodies or antigen-binding fragments of the invention may be of any antibody construction type, for example IgAl, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, F (ab) 2, Fv, scFv, IgGACH2, F (ab ') 2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv) 2, non-depleting IgG, diabody and bivalent antibody . The molecule may also be conjugated to a synthetic molecule. In some embodiments, the invention provides bispecific compounds (eg, bispecific antibodies) comprising the TrkB agonist antibodies or antigen binding fragments described herein.

  In another aspect, the present invention provides polynucleotides encoding heavy and / or light chain variable regions of the antibody or antigen-binding fragment of the present invention, as well as vectors comprising such polynucleotides. In another aspect, the invention provides a pharmaceutical composition or kit comprising a therapeutically effective amount of an antibody or antigen binding fragment as described herein or a polynucleotide or vector expressing said antibody.

  In yet another aspect, the invention provides methods for promoting survival, synaptic function or regeneration of retinal ganglion cells, motor neurons or neurons of the central nervous system (CNS) in a subject. Also provided are methods for treating or preventing a degenerative pathological condition of a subject's eye, a motor neuron disease or a degenerative disease of the central nervous system (CNS). Such therapeutic methods of the invention administer to a subject a pharmaceutical composition comprising a therapeutically effective amount of a TrkB agonist antibody or antigen binding fragment described herein or a polynucleotide or expression vector encoding the antibody. To do. In various embodiments, the pharmaceutical composition can be administered to one or both eyes of a subject. Typically, the pharmaceutical composition to be administered further comprises a pharmaceutically acceptable carrier. A subject suitable for the method may be a subject suffering from or at risk of developing a degenerative disorder of the eye, a motor neuron disease or a degenerative disorder of the central nervous system (CNS). The degenerative disorders suitable for the method of the present invention include, for example, glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion, central retinal vein occlusion, diabetic neuropathy, age-related macular degeneration (AMD) , Anterior ischemic optic neuropathy (AION) or diabetic retinopathy. Examples of motor neuron diseases suitable for treatment with the method of the present invention include, for example, ALS, idiopathic motor neuropathy, hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscle atrophy Disorders (PMA), progressive bulbar palsy (PBP) and pseudobulbar palsy, Bell's palsy or spinal muscular atrophy (SMA). Examples of degenerative diseases of the CNS that can be treated by the method of the present invention include, for example, Alzheimer's disease, Parkinson's disease or Huntington's disease.

  A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and the appended claims.

FIG. 1 shows a scheme for the construction of human TrkB reporter to select TrkB agonist Abs. FIG. 2 shows the activity of several crude scFv-Fc TrkB agonist antibodies in culture supernatant examined by TrkB reporter cell line. FIG. 3 shows the activity of some TrkB agonist IgG antibodies in TrkB reporter cell assay. FIG. 4 shows the selectivity (no effect on TrkA) of the identified TrkB agonist antibodies. FIG. 5 shows that signaling of TrkB agonist Ab resulted in activation of P-PLCγ, P-Akt and P-MAPK in TrkB reporter cells. FIG. 6 shows agonist Ab induced TrkB phosphorylation in cultured mouse cortical neurons. FIG. 7 shows expression of Arc induced by BDNF and TrkB agonist Ab NFAT-85. FIG. 8 shows TrkB phosphorylation induced by agonist antibody NFAT-85 in mouse embryonic stem cells. FIG. 9 shows that the TrkB agonist Ab NFAT-85 suppresses the regression of RGC dendrites. FIG. 10 is an experimental paradigm of efficacy testing of the effects of TrkB agonist antibodies in a rat model of glaucoma. FIG. 11 shows the characteristics of the IOP of the OHT rat model. FIG. 12 shows shawl plot analysis of the effects of TrkB agonist Ab85 and BDNF in OHT rats. FIG. 13 shows the dendritic metrics of the shawl analysis shown in FIG.

I. General Description The present invention is described, in part, with respect to the development by the present inventors of TrkB activating antibodies that have shown utility in the neuroprotective and neuroregenerative treatments of retinal disorders such as glaucoma. The neuroprotective and neuroregenerative activity of BDNF is shown herein by BDNF greatly retarding dendrite regression of RGC and that BDNF can reverse RGC dendrite degeneration. Importantly, the inventors have demonstrated that the TrkB agonist antibodies of the invention show equal or better properties to BDNF as neuroprotective agents of RGCs.

  As detailed herein, we generated the fully human agonist antibody family by selecting rare activating agonist antibodies from combinatorial libraries. In the illustrated test, it was shown that TrkB agonist antibodies can mimic the efficacy of BDNF responding rapidly in the axotomy-cut adult mammalian retina in the maintenance or regeneration of RGC arbors. Specifically, compared to BDNF, the antibody was shown to be potent and selective in both TrkB cell lines and BDNF-responsive primary neuronal cultures. The antibody was also found to be equivalent to BDNF in activating TrkB signaling and also to have cross-species activity (eg, human and mouse). In situ, the antibody was found to be active in retarding and restoring RGC degeneration in adult mouse retinal explant assays. Furthermore, the TrkB agonist Ab showed a regenerative effect on the dendritic tree of retinal ganglion neurons, as assessed by a rat model of hypertensive glaucoma.

Compared to BDNF, the TrkB agonist antibodies of the invention also have biophysical properties that are greatly improved in human subjects over BDNF itself (e.g. unlike BDNF, this antibody is "sticky". Have a much longer half life (eg, better pharmacokinetics). Also, all currently approved treatments for glaucoma are focused on reducing intraocular pressure (IOP) in the anterior chamber of the eye. However, such treatment alone is insufficient to stop the progression of the disease. In contrast, the disease modifying neuroprotective activity of TrkB agonist antibodies disclosed herein may reduce dendrite and cell body atrophy or reduction of RGCs, thus delaying disease progression. In addition, the antibody neural regeneration activity may restore the functional integrity of impaired RGC. According to such a test, according to the present invention, the TrkB tyrosine kinase receptor is specifically activated to survive or regenerate RGC. Provided are monoclonal antibodies and related antigen-binding fragments that promote. Also, in the present invention, such antibody drugs are associated with various neurodegenerative diseases associated with RGC death or degeneration, such as retinal degeneration disorder, or eye diseases mediated by abnormal or impaired Trk B signaling activity. For example, methods are provided for use in therapeutic applications to treat glaucoma.

II. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. The following references give the person skilled in the art a general definition of the many terms used in the present invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1 ^st ed. Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3 ^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1 ^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 ^th ed., 2000). Also, the following definitions are set forth to assist the reader in practicing the present invention.

  The terms "antibody" or "antigen binding fragment" refer to a polypeptide chain (s) exhibiting strong monovalent, bivalent or multivalent binding to a given antigen, epitope (s). Say. Unless otherwise stated, the antibodies or antigen binding fragments used in the present invention may have sequences derived from any vertebrate species, such as camelids, birds or fish species. This can be made using any suitable technique, such as hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Unless otherwise stated, the term "antibody" as used herein is an intact antibody, an antigen binding polypeptide fragment and other designer antibodies as described below or well known in the art (e.g. Serafini). J. Nucl. Med. 34: 533-6 (1993)).

  Intact "antibodies" typically comprise at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) interconnected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes and the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which further define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

Each heavy chain of the antibody is comprised of a heavy chain variable region (V _H ) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C _H1 , C _H2 and C _H3 . Each light chain is comprised of a light chain variable region (V _L ) and a light chain constant region. The light chain constant region is comprised of one domain, C _L. The heavy and light chain variable regions contain binding domains that interact with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune mechanism and the first component (Clq) of the classical complement system.

The V _H and V _L regions of antibodies can be further subdivided into hypervariable regions, also referred to as complementarity determining regions (CDRs), interspersed with more conserved framework regions (FR). Each V _H and V _L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The location of CDR and FR regions and numbering system are described, for example, in Kabat et al. , Sequences of Proteins of Immunological Interest, U. S. Department of Health and Human Services, U.S. S. It is defined by the Government Printing Office (1987 and 1991).

The binding affinity is generally expressed as the equilibrium binding constant or dissociation constant (K _a or K _d respectively), which in turn is the inverse ratio of the dissociation rate constant and the association rate constant (k _off and k _on respectively) is there. Thus, equivalent affinities may correspond to different rate constants, as long as the ratio of rate constants remains the same.

  The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With regard to a specific nucleic acid sequence, conservatively modified variants are nucleic acids encoding amino acid sequences which are identical or essentially identical, or sequences which are essentially identical if the nucleic acid does not encode an amino acid sequence. Say. Because of the degeneracy of the genetic code, any given protein is encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, wherever alanine is specified by a codon, the codon may be altered to any of the corresponding codons described without alteration of the encoded polypeptide. Such nucleic acid mutations are "silent mutations", which are one of the species that are conservatively modified mutations. Also, any nucleic acid sequence herein which encodes a polypeptide indicates any possible silent variation of the nucleic acid. For those skilled in the art, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain functionally identical molecules. It will be recognized. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

  In polypeptide sequences, "conservatively modified variants" refers to variants having conservative amino acid substitutions which are amino acid residues replaced with other amino acid residues having side chains with similar charges. Amino acid residue families having side chains with similar charges are defined in the art. Such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having non-charged polar side chains (eg, For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains Amino acids (eg, threonine, valine, isoleucine) and amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

  The term "contacting" has its ordinary meaning and refers to combining two or more agents (e.g., a polypeptide or a phage), combining an agent with a cell, or combining two different cell populations. . The contacting may be performed in vitro, eg, mixing the antibody and cells, or mixing the antibody population with the cell population in vitro or in growth media. In addition, the contacting may be carried out in cells or in situ, for example, by coexpression of the two polypeptides in the cell and recombinant polynucleotides encoding the two polypeptides in the cells. Alternatively, it may be carried out by contacting in a cell lysate. The contacting may be performed in vivo in a subject, for example, by administering the drug to the subject for delivery of the drug to a target cell.

  The term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody is bound and comprised of one or more amino acid segments. The epitopes may be linear or steric epitopes, and may be continuous or discontinuous. Typically, linear epitopes are contiguous, ie composed of one contiguous amino acid chain. A steric epitope can be discontinuous, ie, composed of two or more noncontiguous amino acid segments that together form an epitope when the antigen is folded. Methods for determining whether an antibody binds the same epitope are known in the art. Epitopes can be defined or mapped by standard methods well known in the art. For example, epitopes can be mapped using an assay such as an ELISA assay, using a peptide library, or with site directed mutagenesis of an antigen (eg, alanine scanning of an antigen).

  As used herein, “binds to the same epitope” with respect to two or more antibodies means that the antibodies compete for binding to the antigen, overlap, or overlap continuous or discontinuous the same amino acid segment It means to bind to. One skilled in the art will understand that the phrase "bind to the same epitope" does not necessarily mean that the antibodies bind to exactly the same amino acid. The exact amino acids to which the antibody binds may be different. For example, a first antibody can bind to a segment of the amoniic acid that is completely encompassed by the amino acid segment to which the second antibody binds. In another example, a first antibody binds to the one or more segments that significantly overlap one or more amino acid segments to which the second antibody binds. For interpretation herein, such an antibody is considered to "bind to the same epitope".

Antibody competition assays can be used to determine if an antibody "binds to the same epitope" as another antibody. Such assays are well known in the art. Typically, an antibody that is known to interact with an epitope by the second antibody under conditions where the second antibody is in excess and all sites are saturated with the first. % Or more, eg 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more of the competitions said that the antibodies bind to the same epitope To indicate that To assess the level of competition between the two antibodies, for example, radioimmunoassays or assays using other labels for antibodies, such as biotin, can be used. For example, the antigen may be combined with a saturating amount of a first antibody or antigen-binding fragment thereof conjugated to a labeled compound (eg, ³ H, ¹²⁵ I or biotin) in the presence of the same amount of a second unlabeled antibody. It can be incubated. The amount of labeled antibody bound to the antigen in the presence of unlabeled blocking antibody is then assessed and compared to binding in the absence of unlabeled blocking antibody. Competition is measured by the percentage change in signal binding in the presence of unlabeled blocking antibody as compared to no blocking antibody. Thus, if 70% inhibition of labeled antibody binding is seen in the presence of blocking antibody as compared to binding in the absence of blocking antibody, there is a 70% competition between the two antibodies. Thus, 70% or more between the first and second antibodies, eg 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or Further reference to competition is that the first antibody has 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85% of the binding of the second antibody to the antigen (or vice versa) , 90%, 95% or more (as compared to antigen binding by the second antibody in the absence of the first antibody). Thus, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more inhibition of the binding of the first antibody to the antigen by the second antibody Indicates that the two antibodies bind to the same epitope. The terms "identical" or "identity" indicate that in the context of two or more nucleic acid sequences or polypeptide sequences, the two or more sequences or subsequences are the same. The two sequences are compared with the greatest match in the comparison window or in one or more of the following sequence comparison algorithms, or in designated areas when measured by manual alignment and visual inspection: When aligned, if the two sequences have the same percentage of amino acid residues or nucleotides in the designated region (ie 60% identity, 65%, 70%, 75%, 80%, It may be 85%, 90%, 95% or 99% identity or, if not specified, in the whole sequence) "substantially identical". Optionally, identity is in a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. Exists.

  Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignments for comparison can be found, eg, in Smith and Waterman, Adv. Appl. Math. 2: 482 c, local homology algorithm of 1970; Needleman and Wunsch, J. et al. Mol. Biol. 48: 443, 1970 homology alignment algorithm; Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988, similarity search method; computer implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI)); It can be carried out by inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively, see Altschul et al. , Nuc. Acids Res. 25: 3389-3402, 1977; and Altschul et al. , J. Mol. Biol. 215: 403-410, 1990.

  As used herein, a degenerative pathological condition or disorder of the eye is characterized by degeneration or death of retinal ganglion cells (RGC), or a disease of the eye associated with said degeneration or death, Or ocular disorder that is mediated by, or associated with, TrkB signaling activity. This includes various clinical features and etiologies. Examples of such disorders include glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion and central retinal vein occlusion.

  Glaucoma is an exemplary degenerative condition of the eye characterized by pathological changes of the optic nerve, visible and corresponding visual field defects in the optic disk, and blindness if untreated. Glaucoma is associated with high intraocular pressure, but other factors are also involved. Glaucoma encompasses the more common types of open angle glaucoma and less common types such as closed angle glaucoma and normal tension glaucoma. The treatment currently used for glaucoma relates to intraocular pressure drop. Medications include transsurface drops or oral dosages, which reduce the occurrence or increase of the outflow of intraocular fluid. However, such drug treatments for glaucoma sometimes have significant side effects, such as headache, fog, allergic reactions, death from cardiopulmonary complications and possible interactions with other drugs. Also, surgical treatments are used, but also have many disadvantages and a lower success rate.

  Motor neuron disease (MND) is any of several nervous system disorders in which motor neurons, which are cells that control the voluntary muscles of the body, are selectively affected. Such diseases include, for example, amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar paralysis (PBP) and pseudobulbar palsy. In some cases, spinal muscular atrophy is also included in this group. Motor neuron disease is neurodegenerative in nature and progressively leads to disability and eventually death. The TrkB agonist antibodies disclosed herein may be used to treat or ameliorate the symptoms of any of such disorders, such as ALS.

  The term "subject" refers to human and non-human animals (especially non-human mammals). The term "subject" is used herein to denote, for example, human or animal subjects in the context of therapeutic and diagnostic methods. Animal subjects include, but are not limited to, animal models of pathological conditions or disorders associated with degeneration or death of retinal ganglion cells, such as mammalian models. Other specific examples of non-human subjects include, for example, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.

  The terms "target", "target molecule" or "target cell" refer to a molecule or biological cell of interest to be analyzed or detected, eg, a eukaryotic cell that modulates its death.

  The terms "treat", "treating", "treatment" and "therapeutically effective" as used herein do not necessarily imply 100% or complete treatment. Rather, there is a varying degree of treatment that is recognized by those skilled in the art as having a potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount of any level of treatment. Furthermore, the treatment provided by the methods of the present invention may include the treatment of one or more pathological conditions or symptoms of the disease to be treated.

  Tropomyosin receptor kinase B (Trk B) is also known as tyrosine receptor kinase B, BDNF / NT-3 growth factor receptor or type 2 neurotrophic receptor tyrosine kinase and in humans is encoded by the NTRK2 gene It is a protein. TrkB is a receptor for brain-derived neurotrophic factor (BDNF). Klein et al. , Cell 61: 647-56, 1990; and Squinto et al. , Cell 65: 885-93, 1991. TrkB is a high affinity catalytic receptor for several "neurotrophins" which are small protein growth factors that induce survival and differentiation of different cell populations. Neurotrophins that activate TrkB are: BDNF (brain derived neurotrophic factor), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3). As such, TrkB mediates multiple effects of such neurotrophic factors, including neuronal differentiation and survival. Studies have shown that activation of the Trk B receptor can lead to downregulation of the KCC dichloride transporter in cells of the CNS.

  A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another polynucleotide segment may be linked so as to bring about the replication of said linked segment. Vectors capable of directing the expression of genes encoding one or more polypeptides are referred to as "expression vectors".

III. TrkB Agonist Antibodies and Related Antigen Binding Molecules The present invention provides antibodies or antigen binding fragments that specifically bind to TrkB (eg, human TrkB) and activate the TrkB signaling pathway. Preferably, the TrkB agonist antibody of the present invention has the same binding specificity as that of the TrkB agonist antibody exemplified herein (eg, antibody NFAT-85 or antibody CRE-30 shown in Table 1). Therefore, according to the present invention, (1) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and SSRDSSRSHHLL (SEQ ID NO: 73); (2) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ARQGASSTSYDY (SEQ ID NO: 48), QSISSY (SEQ ID NO: 49), AAS and QQANSFPVA (SEQ ID NO: 50); ) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56); 4) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKTDSDK (SEQ ID NO: 58) and AIWHSSAV (SEQ ID NO: 56); (5) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 55), AIWHSSAV (SEQ ID NO: 56) (6) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSACV (SEQ ID NO: 59) ) (7) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), ARQLLY (SEQ ID NO: 60), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 61), AIWHSSAV (SEQ ID NO: 56) (8) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ATRVLPAGHF (SEQ ID NO: 62), NIGDKF (SEQ ID NO: 63), YDS and QVWDNSSNQGV (SEQ ID NO: 64); (9) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and NSRDGSGNNV (SEQ ID NO: 69); or (10) GYTFTSYA (SEQ ID NO: No .: 70), INTNTGNP (SEQ ID NO: 71), ASRLAAAGFDY (SEQ ID NO: 72), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56) Included are antibodies or antigen binding fragments having the same binding specificity as those of antibodies having the sequences of CDR1-3 and light chain CDR1-3.

  Some antibodies of the invention can compete with the exemplified antibodies for binding to TrkB. Some antibodies of the invention bind to the same epitope as that recognized by one of the TrkB antibodies exemplified herein. Some antibodies of the invention bind to the same epitope on TrkB with the same or similar affinity as one of the TrkB antibodies exemplified herein. Once bound to TrkB, some antibodies of the invention modulate one or more of TrkB's biological activities in the same or similar manner as with one of the TrkB antibodies exemplified herein. Or may generate a cellular response, such as activation of MAPK, phospholipase C-γ and / or PI3-K signaling pathways.

The antibody of the present invention includes an intact antibody (eg, an IgG1 antibody), an intact antibody fragment or antigen-binding fragment (eg, an antigen-binding portion of an intact antibody), which retains the ability to bind to a cognate antigen. The scFv antibodies exemplified herein are included. Typical intact antibodies interact with the target antigen primarily through amino acid residues that are located within the six complementarity determining regions (CDRs) of the heavy and light chains. The functional TrkB agonist antibody of the present invention has at least one of the sequences of heavy chain CDRs and light chain CDRs that are the same as or substantially identical to the corresponding CDR sequences of the exemplified TrkB agonist antibody Included are antibodies or antigen binding fragments. In some embodiments, TrkB agonists of the invention are antibody fragments or antigen binding molecules derived from the exemplified antibodies. Examples of such antibody fragments include: (i) Fab fragments which are monovalent fragments consisting of V _L , V _H , C _L and C _H1 domains; (ii) two Fab fragments linked by a disulfide bond in the hinge region a bivalent fragment in which F (ab _{') 2} fragments including; (iii) _{V H} and _{C H1} consisting domain Fd fragments; (iv) Fv consisting of the _{V L} and _{V H} domains of an intact one arm of an antibody Fragment; (v) Disulfide stabilized Fv (dsFv) with interchain disulfide bond engineered between structurally conserved framework regions; (vi) single domain antibody (dAb) consisting of _VH or _VL domains (See, eg, Ward et al., Nature 341: 544-546, 1989); and (vii) linear or cyclic pep Single complementarity-determining regions as de (CDR) and the like.

The antibodies of the present invention also encompass single chain antibodies. The term "single-chain antibody" comprises in the polypeptide chain generally V _H and V _L domains linked by a spacer peptide or linker sequence, and further domains or amino acid sequences are amino- and / or carboxyl- A polypeptide that may be contained at the end. For example, a single chain antibody may comprise a tethering segment for linking to a coding polynucleotide. As an example, single chain variable region fragments (scFv) are single chain antibodies. As compared to the V _L and V _H domains of Fv fragments encoded by separate genes, the scFv has two domains (eg, by recombinant methods) linked by a synthetic linker. This allows the pair of _VL and _VH regions to be a single protein chain forming a monovalent molecule. Some of the scFv TrkB agonist antibodies of the invention are listed in Table 1. The V _L and V _H sequences and linking linker sequences of these scFV antibodies are shown in Table 2.

  As exemplified herein, scFv TrkB agonist antibodies may be fused to the Fc domain of IgG to obtain scFv-Fc fusion molecules. Fusion with the Fc-domain results in several valuable biological and pharmacological properties. For example, due to the presence of the Fc domain, interaction with salvage neonatal Fc-receptors, as well as delayed renal clearance due to increased molecular size, the plasma half-life of this hybrid molecule is pronounced It can be increased, which prolongs the therapeutic activity. Furthermore, the addition of the Fc region can also improve the solubility and stability of the fused partner, allowing cost-effective facile purification by protein-G / A affinity chromatography at the time of manufacture. The scFv antibody fragment and the fusion partner, such as a hybrid molecule or fusion molecule comprising an Fc domain, can be readily produced according to standard recombinant techniques, routinely practiced protein synthesis methods or protocols described herein. .

The antibodies of the invention also encompass single domain antigen binding units having a camelid backbone. Camelids include camels, llamas and alpacas. Camelids make functional antibodies free of light chains. The heavy chain variable (V _H ) domain spontaneously folds and functions independently as an antigen binding unit. The binding surface has only three CDRs, whereas the classical antigen binding molecule (Fab) or single chain variable fragment (scFv) contains six CDRs. With camelid-derived antibodies, binding affinities comparable to those of conventional antibodies can be obtained.

In addition to the TrkB agonist antibodies exemplified herein initially identified by functional screening, various other antibodies or antigen binding fragments of the invention may also be generated by enzymatic or chemical modification of intact antibodies. Or can be synthesized de novo using recombinant DNA methodology or can be identified using a phage display library. As an illustration, specific scFv antibodies shown in Table 1 can be readily converted to scFv-Fc fusions by standard recombinant techniques, for example, by cloning into the pFUSE IgG backbone vector described herein. Can. All additional methods that can be used to generate other TrkB agonist antibodies or antigen binding molecules of the invention are well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffle libraries (eg, McCafferty et al., Nature 348: 552-554, 1990; and US Pat. No. 4 , 946, 778). In particular, scFv antibodies are disclosed, for example, in Bird et al. , Science 242: 423-426, 1988; and Huston et al. , Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988. They can be obtained using the method described in U.S. Pat. Fv antibody fragments can be produced as described in Skerra and Pluckthun, Science 240: 1038-41, 1988. Disulfide stabilized Fv fragments (dsFv) are described, for example, in Reiter et al. , Int. J. Cancer 67: 113-23 (1996). Similarly, single domain antibodies (dAbs) are described, for example, by Ward et al. , Nature 341: 544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93: 6280-85, 1996, which can be made by various methods. Single domain antibodies derived from camelids are well known in the art, for example, Dumuulin et al. , Nat. Struct. Biol. 11: 500-515, 2002; Ghahroudi et al. , FEBS Letters 414: 521-526, 1997; and Bond et al. , J. Mol. Biol. 332: 643-55, 2003 can be used. Also, other types of antigen binding fragments (eg, Fab, F (ab ') ₂ or Fd fragments) can be readily generated using routinely practiced methods of immunology. See, eg, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.

  In some embodiments, the antibody or antigen binding fragment of the invention is a heavy chain CDR1, CDR2 and CDR3 sequence and / or a light chain CDR1, CDR2 and CDR3 substantially identical to that of the antibodies shown in Table 1 Have an array of The sequences of the light chain and heavy chain CDRs of the exemplified antibodies are all shown in Table 2. In some of such embodiments, the antibody or antigen-binding fragment comprises: (1) SEQ ID NO: 46-49, AAS and SEQ ID NO: 50; (2) SEQ ID NO: 51-56; (3) Sequence Nos. 51 to 53, 57, 58 and 56; (4) SEQ ID NOs: 51 to 53, 57, 55 and 56; (5) SEQ ID NOs: 51 to 55 and 59; (6) SEQ ID NO: 51, 52, 60, 57, 61 and 56; (7) SEQ ID NO: 46, 47, 62, 63, YDS and SEQ ID NO: 64; (8) SEQ ID NO: 65 to 68, GKN and SEQ ID NO: 69; (9) Sequence Or (10) the sequences of heavy chain CDRs 1 to 3 and the sequences of light chain CDRs 1 to 3 substantially identical to SEQ ID NO: 65 to 68, GKN and SEQ ID NO: 73; Have. In some such embodiments, the percentage of sequence identity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or It may be at least 99% or even 100%. Thus, some antibodies or antigen-binding fragments of the invention have the sequences of heavy chain CDRs 1 to 3 and / or light chain CDRs 1 to 3 that are at least 90% identical to the corresponding CDRs of one of the exemplified antibodies, respectively. Have. Some such antibodies include heavy chain CDR sequences and / or light chain CDR sequences that are each at least 95% identical to the corresponding CDRs of the exemplified antibodies. In some other antibodies or antigen binding fragments of the invention, the heavy chain CDR sequences and / or the light chain CDR sequences are each at least 95% identical to the corresponding CDR of one of the exemplified antibodies. In some embodiments, the sequences of CDR1 to CDR3 of the heavy chain of the antibody and CDR1 to CDR3 of the light chain are (1) SEQ ID NO: 46 to 49, AAS and SEQ ID NO: 50; (2) SEQ ID NO: (3) SEQ ID NOS: 51-53, 57, 58 and 56; (4) SEQ ID NOS: 51-53, 57, 55 and 56; (5) SEQ ID NOS: 51-55 and 59; ) SEQ ID NO: 51, 52, 60, 57, 61 and 56; (7) SEQ ID NO: 46, 47, 62, 63, YDS and SEQ ID NO: 64; (8) SEQ ID NO: 65 to 68, GKN and Sequences (9) SEQ ID NOs: 70-72 and 54-56; or (10) SEQ ID NOs: 65-68, GKN and identical to the sequence shown in SEQ ID NO: 73.

  In another embodiment, the antibody or antigen-binding fragment that specifically binds human TrkB is (a) SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 Or a heavy chain variable region substantially identical to 38, (b) SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 31, 33, 35, 37 or 39 substantially identical Or (c) both the heavy chain variable region of (a) and the light chain variable region of (b). In some embodiments, the antibody comprises both the heavy chain of (a) and the light chain of (b). In some embodiments, the antibody or antigen binding fragment is (a) any of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38. Any of heavy chain variable domains having at least 90% identity with one, (b) SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 31, 33, 35, 37 and 39 Or a light chain variable domain having at least 90% sequence identity with one or both of the heavy chain of (c) (a) and the light chain of (b). In some embodiments, the antibody or antigen binding fragment comprises at least one of SEQ ID NO: 15, 17, 19, 21, 23, 25, 25, 29, 31, 31, 33, 35, 37 and 39 Contains light chain variable domains with 90% identity. In some such embodiments, the percentage of sequence identity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or It may be at least 99% or even 100%. In some embodiments, the light chain variable domain comprises at least 95 of any one of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 31, 33, 35, 37 and 39. % Identity. In some embodiments, the light chain variable domain is 100% with any one of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 31, 33, 35, 37 and 39. Have the same identity. In some other embodiments, the antibody or antigen binding fragment is any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 And heavy chain variable domains having at least 90% identity. In other embodiments, the percentage of identity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or even 100 % Can be. In some embodiments, the heavy chain variable domain comprises at least 95 of any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 % Identity. In some embodiments, the heavy chain variable domain is 100% with any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 Have the same identity.

  In addition to the above heavy chains, the antibodies or antigen binding fragments of the invention may further comprise light chains selected from a Fab library using sequential shuffling of naive chains. Similarly, in addition to the light chain described above, the antibodies of the invention may further comprise a heavy chain selected from a Fab library using sequential shuffling of the naive chain. In some embodiments, an antibody or antigen-binding fragment of the invention is any of the heavy chains described herein (eg, SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, The heavy chains shown in 30, 32, 34, 36 and 38) may be included in combination with any suitable light chain, such as those exemplified herein. Similarly, the antibody may be any suitable one of the light chains described above (eg, the light chains shown in 15, 17, 19, 21, 23, 25, 25, 29, 31, 31, 33, 35, 37 and 39). Heavy chains, such as in combination with those exemplified herein. For example, in some preferred embodiments, the antibodies are SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: : 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37 or SEQ ID NO: It comprises heavy chain variable region sequences and light chain variable region sequences which are at least 90% identical to 38 and 39, respectively. In some embodiments, the antibody comprises SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, SEQ ID NOs: 30 and 31, SEQ ID NOs: 32 and 33, SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37 or SEQ ID NOs: 38 and 39 The heavy chain sequence and the light chain sequence respectively shown in In its various embodiments, the percent identity (%) of the peptide sequences may be calculated, for example, as 100 × [(identical position) / min (TGA, TGB)], where TGA and TGB are Alignment is the sum of the number of residues of the peptide sequences A and B being aligned and the position of the internal gap, the alignment is such that TGA and TGB are minimized. See, eg, Russell et al, J. Mol. Biol. , 244: 332-350 (1994).

  An antibody or antigen-binding fragment of the invention is any that specifically recognizes or binds to the extracellular domain of TrkB (e.g., human TrkB) with the same specificity as that of the TrkB agonist antibodies (Table 1) exemplified herein. Antibodies, such as full-length antibodies or antibody fragments. The TrkB antibodies of the invention are preferably monoclonal. The antibody may be a recombinant antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Furthermore, the antibody may be of any isotype, including but not limited to IgA, IgD, IgE, IgG or IgM. Thus, for example, the antibody may be any IgA, such as IgAl or IgA2 or any IgG, such as IgG1, IgG2, IgG3, IgG4 or synthetic IgG. In addition, the antibody may be any antibody fragment having specificity for the extracellular domain of human TrkB, such as F (ab) 2, Fv, scFv, IgGACH2, F (ab ') 2, scFv2CH3, Fab, VL, VH, It may be scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv) 2, diabodies and bivalent antibodies. The antibody may be any modified or synthetic antibody, such as, but not limited to, non-depleting IgG antibodies, CAR or other Fc or Fab variants of antibodies.

  In some embodiments, the invention provides antibodies or antigen binding fragments that are conservatively modified variants of the anti-TrkB antibodies exemplified herein. Typically, the variable regions of such variants have an amino acid sequence that is identical to one of these exemplified sequences except for conservative substitutions of one or more amino acid residues. In some embodiments, the antibody or antigen-binding fragment of the invention specifically binds to TrkB and (a) from SEQ ID NOs: 46-48, 51-53, 60, 62, 65-67 and 70-72 And / or (b) SEQ ID NOs: 49, 50, 54-59, 61, 63, 64, 68, 69 and 73 and the sequences AAS, YDS and GKN And at least one LCDR having an arrangement selected from the group consisting of The present invention also provides a TrkB agonist antibody that specifically binds to TrkB and comprises one or more variants of the sequences of the aforementioned CDRs or the sequences of substantially identical CDRs. The sequences of the variant CDRs of such an antibody are within a sequence selected from the group consisting of 1, 2 or 3 substitutions, insertions, deletions or combinations thereof of SEQ ID NO: 46 to 73 and the sequences AAS, YDS and GKN. It can be included in For example, a recombinant chimeric or humanized antibody (or a fragment thereof) may comprise one, two, three, four, five or six of the sequences of the aforementioned CDRs.

  In some embodiments, the TrkB agonist antibody or antigen-binding fragment of the invention comprises sequences of three CDRs of the same light or heavy chain of the antibodies of Table 1. These include, for example, (1) SEQ ID NO: 49, AAS and SEQ ID NO: 50, (2) SEQ ID NOs: 54 to 56, (3) SEQ ID NOs: 57, 58 and 56, (4) SEQ ID NO: 57 , 55 and 56, (5) SEQ ID NOs: 54, 55 and 59, (6) SEQ ID NOs: 57, 61 and 56, (7) SEQ ID NO: 63, YDS and SEQ ID NO: 64, (8) SEQ ID NO: 68, GKN and SEQ ID NO: 69 and (9) SEQ ID NO: 68, GKN and light chain CDRs shown in SEQ ID NO: 73; or (1) SEQ ID NO: 46 to 48, (2) SEQ ID NO: 51 to 53, (3) SEQ ID NOs: 51, 52, 60 (4) SEQ ID NOs: 46, 47 and 62 (5) SEQ ID NOs: 65 to 67 and (6) heavy chain CDRs shown in SEQ ID NOs: 70 to 72 Be In some embodiments, the TrkB agonist antibody or antigen-binding fragment of the invention comprises the sequences of the six CDRs of the same antibody, for example (1) SEQ ID NO: 46-49, AAS and SEQ ID NO: 50 (Antibody CRE- 6); (2) SEQ ID NO: 51-56 (antibody CRE-30 or CRE-83); (3) SEQ ID NO: 51-53, 57, 58 and 56 (antibody CRE-31 or CRE-53); 4) SEQ ID NO: 51-53, 57, 55 and 56 (antibody CRE-39 or NFAT-79); (5) SEQ ID NO: 51-55 and 59 (antibody CRE-87); (6) SEQ ID NO: 51 52, 60, 57, 61 and 56 (antibody CRE-93); (7) SEQ ID NOs: 46, 47, 62, 63, YDS and SEQ ID NO: 64 (antibody NFAT-27); Nos. 65-68, GKN and SEQ ID NO: 69 (antibody NFAT-40); (9) SEQ ID NOs: 70-72 and 54-56 (antibody NFAT-44); or (10) SEQ ID NO: 65-68, GKN and SEQ ID NO: 73 (antibody NFAT-85).

  In some embodiments, the present invention provides an antibody or antigen-binding fragment having an avidity of about 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 400 nM or less, 300 nM or less, or 200 nM or less of TrkB relative to TrkB. Do. In some embodiments, the antibody or antigen binding fragment binds to TrkB with an avidity of about 100 nM or less, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, or about 5 nM or less. In some embodiments, the antibody or antigen binding fragment is about 1 nM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less for TrkB. Combine at the following avidity: Avidity can be measured using techniques known in the art, such as ELISA or surface plasmon resonance.

  The antibodies of the invention can be produced by any suitable technique, for example using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, antibodies are produced using a mammalian expression system. An example of a specific procedure for producing the antibody or antigen-binding fragment of the present invention is exemplified herein. In some embodiments, the antibodies or antigen binding fragments of the invention can be made using any suitable non-eukaryotic expression system, such as a bacterial expression system. The bacterial expression system may be a fragment such as F (ab) 2, Fv, scFv, IgGACH2, F (ab ') 2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc ( It can be used to make scFv) 2 and diabodies. Techniques for altering DNA coding sequences to produce such fragments are known in the art.

  The antibodies or antigen binding fragments of the invention may be conjugated to synthetic molecules using any type of suitable conjugation. It can be conjugated to synthetic molecules using recombinant manipulation and incorporation of selenocysteine (eg, as described in US Pat. No. 8,916,159, issued Dec. 23, 2014). Other conjugation methods may include covalent coupling with natural or engineered lysine side chain amines or cysteine side chain thiols. For example, Wu et al. , Nat. Biotechnol, 23: 1 137-1 146 (2005). Synthetic molecules can be any molecule, for example, one that targets a molecule of interest (eg, a cell surface receptor). In some embodiments, synthetic molecules for conjugation to antibodies are proteins (eg, antibodies) or RNA or DNA aptamers. In some embodiments, TrkB agonist antibodies (eg, scFv molecules) of the invention are present as components of a bispecific or multispecific molecule. Typically, the bispecific or multispecific compounds of the invention are covalently or noncovalently attached to at least one other binding moiety that specifically recognizes a target of interest (eg, TrkC or TNFα) It comprises a conjugated TrkB agonist antibody or antigen binding fragment. The binding moiety may be a molecule of any chemical nature, such as another antibody or antigen binding fragment, a polypeptide or peptide agent, or a small molecule agent. In some embodiments, the multispecific molecule of the invention comprises a TrkB agonist antibody and another antibody or antigen binding fragment well known in the art, such as the TNFα antibody adalimumab exemplified herein. It is a bispecific antibody. In various embodiments, the other binding portion of such bispecific or multispecific molecule is edrecolomab, capromab (capromab), ibritumomab, blinatumomab, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, Silutuximab, traibizumab, alemtuzumab, omalizumab, bevacizumab, natalizumab, ranibizumab, eculizumab, seltrizumab, pertuzumab, povintuzumab, etozumab, edulzumab, a patient, a uch Ipilimumab, laxibacubab, nivolumab, rhamsi Mab, Arirokumabu, Daratsumumabu, evolocumab may be Neshitsumumabu and secukinumab. In some other embodiments, the multispecific molecule of the invention is a bispecific small molecule-antibody conjugate. Compared to monospecific antibodies, bispecific compounds may result in enhanced drug efficacy and / or desired targeting manipulations.

IV. Polynucleotides, Vectors and Host Cells for Making TrkB Antibodies According to the invention, identical to a sequence encoding a polypeptide comprising a segment or domain of an antibody chain or an antigen binding molecule as described herein, or Or provide a substantially purified polynucleotide (DNA or RNA) complementary to the sequence. In some embodiments, the polynucleotides of the invention encode sequences of heavy and / or light chain domains shown in Table 1. When expressed in an appropriate expression vector, polypeptides encoded by such polynucleotides can exhibit antigen binding ability. Also provided herein are polynucleotides encoding at least one CDR region, usually all three CDR regions, of the heavy or light chains of the antibodies described herein. Some other polynucleotides encode all or substantially all of the heavy and / or light chain variable region sequences of the exemplified antibodies. For example, a portion of such a polynucleotide may be any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 Heavy chain variable region amino acid sequence and / or light chain shown in any one of SEQ ID NOs: 15, 17, 19, 21, 23, 23, 25, 27, 29, 31, 31, 33, 35, 37 or 39 It encodes the amino acid sequence of the variable region. Because of the degeneracy of the code, each of the amino acid sequences of immunoglobulins is encoded by various nucleic acid sequences.

  The polynucleotide of the present invention may encode only the sequence of the variable region of the exemplified antibody. The polynucleotide may also encode both the variable region and the constant region of the antibody. The polynucleotide sequence of a portion of the nucleic acid of the invention is shown in any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 Encodes a sequence of a mature heavy chain variable region that is substantially identical (eg, at least 80%, 90% or 99%) to that of the mature heavy chain variable region. Another part of the polynucleotide sequence is the mature light set forth in any one of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 25, 29, 31, 31, 33, 35, 37 or 39 It encodes the sequence of a mature light chain variable region which is substantially identical to that of the chain variable region. A portion of the polynucleotide sequence encodes a polypeptide comprising the variable regions of both heavy and light chains of one of the exemplified antibodies. Some other polynucleotides encode two polypeptide segments that are substantially identical to the heavy chain and light chain variable regions of one of the exemplified antibodies, respectively.

  The polynucleotide sequences can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of existing sequences encoding the exemplified functional antibodies (eg, the sequences described in the examples below). Direct chemical synthesis of nucleic acids can be performed by methods known in the art, for example, Narang et al. , Meth. Enzymol. 68: 90, 1979, the phosphotriester method; Brown et al. , Meth. Enzymol. 68: 109, 1979, the phosphodiester method; Beaucage et al. , Tetra. Lett. 22: 1859, the diethyl phosphoramidite method of 1981; and the solid support method of US Pat. No. 4,458,066. The introduction of mutations into polynucleotide sequences by PCR is described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H., et al. A. Erlich (ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al. , Nucleic Acids Res. 19: 967, 1991; and Eckert et al. , PCR Methods and Applications 1:17, 1991.

  The present invention also provides expression vectors and host cells for producing the functional antibodies described herein. Specific examples of lentiviral vectors for expressing the antibodies are described in the following examples. Also, various other expression vectors can be used to express a polynucleotide encoding the functional antibody chain or binding fragment. Both viral and non-viral expression vectors can be used to produce the antibody in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with an expression cassette for expressing proteins or RNA) and human artificial chromosomes (eg Harrington et al., Nat. Genet. 15: 345, 1997). For example, non-viral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (eg, human) cells include pCEP4, pREP4, pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Other useful non-viral vectors include Sleeping Beauty, PiggyBack and other transposon systems. Useful viral vectors include lentivirus or other retrovirus, adenovirus, adeno-associated virus, herpes virus based vector, SV40, papilloma virus, HBP Epstein-Barr virus based vector, vaccinia virus vector and Semliki Forest virus (SFV) is mentioned. Brent et al. Smith, Annu. Rev. Microbiol. 49: 807, 1995; and Rosenfeld et al. , Cell 68: 143, 1992.

  The choice of expression vector depends on the host cell for which the vector is intended to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (eg, an enhancer) operably linked to the polynucleotide encoding the functional antibody chain or fragment. In some embodiments, an inducible promoter is used to suppress expression of the inserted sequence outside of the induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoter or heat shock promoter. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population against coding sequences whose expression products are more tolerable by the host cell. Also, in addition to the promoter, other regulatory elements may be required or desirable for efficient expression of functional antibody chains or fragments. Such elements typically include the ATG initiation codon and adjacent ribosome binding sites or other sequences. Also, the efficiency of expression can be enhanced by including an appropriate enhancer in the cell line used (eg Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153: 516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

  The expression vector may also supply a secretion signal sequence position for forming a fusion protein with the polypeptide encoded by the sequence of the functional antibody to be inserted. Most often, the sequence of the functional antibody to be inserted is ligated to the signal sequence prior to inclusion in the vector. Also, in some cases, the vector used to receive sequences encoding the variable domains of the light and heavy chains of functional antibodies also encodes the constant region or a portion thereof. In such vectors, the variable region can be expressed as a fusion protein having a constant region, which results in the production of an intact antibody or fragment thereof. Typically, such constant regions are human.

  Host cells for housing and expressing functional antibody chains can be either prokaryotic or eukaryotic systems. In some preferred embodiments, mammalian host cells are used to express and produce antibody polypeptides of the invention. For example, the cells can be either hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring foreign expression vectors. Such include any normal dead or normal or abnormal immortal animal or human cells. In addition to the cell lines exemplified herein, several other suitable host cell lines capable of secreting intact immunoglobulins are also known in the art. These include, for example, CHO cell lines, various HEK 293 cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally described, for example, in Winnacker, From Genes to Clones, VCH Publishers, N .; Y. , N. Y. , 1987. Expression vectors for mammalian host cells may include expression control sequences such as replication origins, promoters and enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. . Such expression vectors usually contain a promoter derived from a mammalian gene or mammalian virus. Suitable promoters may be constitutive, cell type specific, phase specific and / or modulatable or regulatable. Useful promoters include, but are not limited to, the EF1 alpha and human UbC promoters exemplified herein, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone inducible MMTV promoter, SV40 promoter, MRP pol III promoter, constitutive MPSV promoter These include the tetracycline inducible CMV promoter (eg, the human immediate early CMV promoter), the constitutive CMV promoter, as well as promoter-enhancer combinations known in the art.

  Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transformation is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts (generally Sambrook et al., Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor Press (3rd edition, 2001)). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic flight method, virosome, immunoliposome, polycation: nucleic acid conjugate, naked DNA, artificial virion And fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88: 223, 1997), drug-enhanced uptake of DNA, as well as ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is often desired. For example, a cell line that stably expresses the antibody chain or binding fragment can be prepared using the expression vector of the present invention comprising a viral origin of replication or an endogenous expression element and a selectable marker gene. After introduction of the vector, cells can be cultured in rich medium for 1 to 2 days, after which the medium is replaced with selective medium. The purpose of the selectable marker is to confer resistance for selection, the presence of which allows growth in selective media of cells which successfully express the introduced sequences. Stably transfected resistant cells can be grown using tissue culture procedures appropriate to the cell type.

V. Therapeutic Applications The TrkB antibodies disclosed herein can be used in a variety of therapeutic applications. Some methods of the invention are directed to induce activation of the TrkB signaling pathway in TrkB-expressing cells, such as retinal ganglion cells, or to enhance the signaling activity of the pathway. Such methods can be used for in vitro or in vivo modification of cells. In such methods, a therapeutically effective amount of the TrkB agonist of the present invention is typically contacted with the cells. Some other methods of the invention relate to promoting regeneration or survival of retinal ganglion cells in a mammalian subject. For example, such methods can be used to restore, improve or maintain the visual function of the eye of a subject affected by or at risk of developing a degenerative pathological condition of the eye. Some other methods of the invention relate to the treatment or prevention of a subject having or at risk of developing a degenerative pathological condition of the eye. In such methods, a pharmaceutical composition comprising a therapeutically effective amount of a TrkB agonist of the present invention is administered to a subject. Also, in addition to pharmaceutical compositions comprising the TrkB antibodies of the invention, the therapeutic applications of the invention may also use the polynucleotides or vectors discussed herein that encode and express the antibodies. For example, compositions having the antibody-encoding polynucleotides or vectors of the invention can be used in gene therapy for various neurodegenerative diseases as described herein.

  Many ocular degenerative conditions are suitable for therapeutic or prophylactic treatment with the therapeutic methods of the invention. Such include any eye disease or disorder characterized by or associated with degeneration or death of retinal ganglion cells (RGC). Examples of such diseases or pathological conditions include, for example, glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion and central retinal vein occlusion. Other ocular disorders that may be suitable for treatment with the therapeutic antibody composition of the invention include, for example, diabetic neuropathy, macular malady including both wet and dry age-related macular degeneration (AMD). Degeneration, anterior ischemic optic neuropathy (AION), ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinitis pigmentosa and retinal degeneration.

  In some embodiments, the present invention improves TrkB signaling activity in cells expressing TrkB (eg, retinal ganglion cells) by contacting the cells with an antibody or antigen binding fragment of the present invention. Provide a way to In related embodiments, the TrkB agonists of the invention may be used to promote survival or regeneration of retinal ganglion cells in a subject. In such embodiments, the functional integrity of retinal ganglion cells may be maintained or restored as a result of the update of TrkB signaling activity induced by the administered TrkB agonist drug. In various embodiments, the TrkB agonist antibody or antigen binding fragment to be administered may be a naked (non-conjugated) molecule, conjugated to a synthetic molecule, such as a targeting moiety or another therapeutic agent. It may be an antibody molecule. The method may be used to upregulate TrkB signaling in cells in vitro, or in a subject (ie in vivo). The contacted TrkB-expressing cell is, for example, a cell culture of a disorder associated with abnormal or impaired TrkB signaling activation or signaling activity or a disease characterized by degeneration or death of retinal ganglion cells. It may be present in or in animal models.

  In some other embodiments, the present invention provides methods for treating a subject in need of enhanced TrkB signaling. Such subjects may have a disease associated with impaired TrkB signaling activity or a disease that can be treated by enhancement of TrkB signaling, such as motor neuron disease such as amyotrophic lateral sclerosis (ALS). Or a subject suspected of having or at risk of developing the disease. In some embodiments, the present invention provides methods for promoting motor neuron survival, regeneration and synaptic function. Such methods include various motor neuron diseases such as ALS, idiopathic motor neuropathy, hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive It can be used to treat subjects afflicted with bulbar palsy (PBP) and pseudobulbar palsy, Bell's palsy or spinal muscular atrophy (SMA). In some other embodiments, the invention provides methods for promoting survival, regeneration and synaptic function of neurons of the central nervous system (CNS). Such methods can be readily used to treat a subject suffering from a degenerative disorder of the CNS. Many CNS degenerative diseases or disorders are suitable for treatment with the therapeutic composition of the present invention. These include, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease and Tourette's response group.

  Some treatment methods of the invention specifically relate to the treatment of subjects having or at risk of developing an ocular disorder characterized by degeneration or death of retinal ganglion cells. These include some of the degenerative pathological conditions of the eye, such as glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion, central retinal vein occlusion, diabetic neuropathy, wet type And macular degeneration including both age-related and dry age-related macular degeneration (AMD), anterior ischemic optic neuropathy (AION) and diabetic retinopathy.

  In general, the various therapeutic methods described herein comprise a therapeutically effective amount of an isolated or purified TrkB agonist antibody or antigen-binding fragment (or polynucleotide or vector expressing said antibody) of the present invention in a subject. Administering a pharmaceutical composition comprising The antibody has the same binding specificity as any of the anti-TrkB agonist antibodies of the invention described herein in Table 1 or derivatives thereof, such as, for example, those derived from antibody NFAT-85 and / or NFAT-85. It may be an antibody possessed. Thus, the antibody administered may be a synthetic antibody, a chimeric antibody, a humanized antibody or a fully human antibody or an antigen binding fragment. This may be F (ab) 2, Fv, scFv, IgGACH2, F (ab ') 2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv or (scFv) 2. In some embodiments, the method comprises administering an IgG, scFv, dsFv, F (ab ') 2, diabody or bivalent antibody. In some embodiments, the antibody or antigen binding fragment administered is another therapeutic agent (eg, a known drug or compound for treating a degenerative pathological condition of the eye) or other agent (eg, , A targeting agent).

  The TrkB agonist antibodies of the invention may be used in combination with other known agents to enhance TrkB signaling and to treat degenerative pathological conditions of the eye. Such known TrkB agonist compounds include, for example, neurotrophic factor BDNF and neurotrophic factor mimics. Specific examples of known TrkB agonist compounds include, for example, L-783, 281 adenosine, CGS 21680, and the like. For example, Pollack et al. Curr. Drug Targ-CNS and Neurol. See Disorders 1: 59-80 2002. Also, TrkB agonist compounds can be small molecules that mimic critical regions of neurotrophin. For example, the small molecule can be a mimic of the BDNF β-turn loop. Examples of such small molecule mimetics that can be used according to the invention are described, for example, in US Patent Application Publication No. 2007/0060526. Preferably, the TrkB agonist antibody and other known TrkB agonist compounds used in the methods of the present invention are selective for TrkB and are those that activate TrkB to a greater extent than TrkA or TrkC. In some embodiments, the agent used is one that is specific for TrkB and does not activate TrkA or TrkC.

  In some therapeutic applications, the TrkB agonist antibodies of the invention may be used in combination with other known medications or regimens to treat optic neuropathy or retinal degenerative disorders such as glaucoma. For example, the antibody can be used with any existing IOP-lowering drug. Such known drugs include, for example, prostaglandin analogs such as xalatan (Latanoprost) and Lumigan (Vimatroprost), beta-blockers such as timolol, betaxolol and methipranol. In addition to therapeutic IOP-lowering drugs, there are many approved drainage devices that can be used in combination with the antibodies of the invention.

  In some embodiments, the TrkB agonist antibody or antigen binding molecule of the invention may be conjugated to a targeting moiety when used in the treatment methods described herein. The targeting moiety may be a protein or peptide that directs localization to a specific part of the body, such as the brain or a compartment thereof. In some embodiments, TrkB antibody agonists can be conjugated or fused to a brain targeting moiety. The brain targeting moiety may be attached covalently (e.g., directly, by translational fusion, or directly or by chemical attachment either via a spacer molecule (which may be cleavable)), It may be attached non-covalently (eg, by a reversible interaction such as avidin, biotin, protein A, IgG, etc.). Brain delivery of TrkB agonists may be enhanced by brain targeting moieties conjugated to TrkB antibody agonists of the invention. Several agents can be used as brain targeting moieties in the practice of the present invention. These include polypeptides or antibody fragments capable of delivering fusion proteins or therapeutic agents across the blood-brain barrier (BBB). As an example of such an agent, single domain antibody FC5 (Abulrob et al. J. Neurochem. 95, 1201-1214, 2005); monoclonal antibody mAB 83-14 against human insulin receptor (Pardridge et al. Pharmacol. Res. 12 , 807-816, 1995); B2, B6 and B8 peptides which bind to human transferrin receptor (hTfR) (Xia et al. J. Virol. 74, 11359-11366, 2000); OX26 monoclonal antibody against transferrin receptor ( Pardridge et al. J. Pharmacol. Exp. Ther. 259, 66-70, 1991); and as described in U.S. Patent No. 6,306,365. Kano polypeptide.

VI. Pharmaceutical Compositions and Combinations The present invention provides methods of using the TrkB agonists of the present invention in the manufacture of a medicament for treating ocular degenerative disorders, such as glaucoma. The TrkB agonists of the present invention may be administered alone to a subject in need of treatment or amelioration of such a pathological condition. However, administration of a pharmaceutical composition comprising a TrkB agonist and a pharmaceutically acceptable carrier is more preferred. Examples of TrkB agonists of the present invention that may be used in pharmaceutical compositions include any of the TrkB agonist antibodies or antigen binding molecules described herein. Exemplary compositions are SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 25, SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, SEQ ID NOs: 30 and 31, SEQ ID NOs: 32 and 33, SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37 or SEQ ID NOs: 38 and 39 One or more of the antibodies having mature heavy and light chain variable domain sequences. In some embodiments, the antibody comprises SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, SEQ ID NOs: 30 and 31, SEQ ID NOs: 32 and 33, SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37 or SEQ ID NOs: 38 and 39 The heavy chain sequence and the light chain sequence respectively shown in Other antibodies suitable for the pharmaceutical composition of the present invention include the mature chain sequences shown in SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38. And / or those having the sequence of the mature light chain shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.

  A further exemplary therapeutic composition of the invention comprises an antibody having 1, 2, 3, 4, 5 or 6 CDRs selected from the group consisting of SEQ ID NO: 46-73 and the sequences AAS, YDS and GKN. It may be included. However, in some embodiments, the antibody comprises the sequence of three CDRs of the same light or heavy chain as shown and exemplified in Table 1. In some embodiments, the pharmaceutical composition comprises the sequences of the six CDRs of the same antibody as exemplified in Table 1, eg, (1) SEQ ID NO: 46-49, AAS and SEQ ID NO: 50 (antibody CRE-6) (2) SEQ ID NO: 51 to 56 (antibody CRE-30 or CRE-83); (3) SEQ ID NO: 51 to 53, 57, 58 and 56 (antibody CRE-31 or CRE-53); (4) SEQ ID NOs: 51-53, 57, 55 and 56 (antibody CRE-39 or NFAT-79); (5) SEQ ID NOs: 51-55 and 59 (antibody CRE-87); (6) SEQ ID NOs: 51, 52 , 60, 57, 61 and 56 (antibody CRE-93); (7) SEQ ID NO: 46, 47, 62, 63, YDS and SEQ ID NO: 64 (antibody NFAT-27); (8) SEQ ID NO: 65 to 68, GK And SEQ ID NO: 69 (antibody NFAT-40); (9) SEQ ID NOs: 70-72 and 54-56 (antibody NFAT-44); or (10) SEQ ID NO: 65-68, GKN and SEQ ID NO: 73 ( Antibodies include antibodies having NFAT-85). Yet another exemplary pharmaceutical composition comprises dsFv fragments, which may optionally include one or more modifications to the amino acid sequence, as would be understood by one skilled in the art.

  Also provided by the present invention is a pharmaceutical combination product, such as a kit. Such a pharmaceutical combination product is a TrkB agonist of the present disclosure, which comprises the active agent in free form or in the form of a composition, one or more inactive agents or other ingredients, and instructions for the administration of the agent. It can be. In some embodiments, the therapeutic kit of the invention comprises one or more doses of a TrkB agonist (eg, antibody NFAT-85) present in a pharmaceutical composition as described herein; said pharmaceutical composition The device may comprise suitable devices for intravitreal injection of and an instruction manual detailing a suitable subject and a protocol for performing the injection.

  Pharmaceutical compositions comprising TrkB agonist antibodies can be prepared in various forms. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, ampoules Drops or injectable solutions, and also preparations with prolonged release of the active compound. The antibody composition of the present invention may also be prepared in the form of a surfactant and administered to a subject. The pharmaceutical compositions of the invention are prepared according to standard protocols well known in the art, for example, Remington: The Science and Practice of Pharmacy, Gennaro (ed.), Lippincott Williams & Wilkins (20th ed., 2003). It can be done. The pharmaceutical composition typically comprises an effective amount of TrkB agonist antibody sufficient to alleviate or ameliorate the symptoms of degenerative eye disorders. In addition to TrkB agonist antibodies, the pharmaceutical composition may also include certain pharmaceutically acceptable carriers that enhance or stabilize the composition, or facilitate preparation of the composition. For example, TrkB agonist antibodies can be complexed with carrier proteins such as ovalbumin or serum albumin to enhance stability or pharmacological properties prior to administration. In addition, excipients and additives and / or adjuvants in various forms of pharmaceutical compositions such as disintegrants, binders, coatings, swelling agents, lubricants, flavoring agents, sweeteners, and the like Elixirs may be included, including inert diluents commonly used in the art (such as purified water).

  Pharmaceutically acceptable carriers are determined, in part, by the specific composition to be administered as well as by the specific method used to administer the composition. Also, the carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not harmful to the subject. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, eg, oral, sublingual, rectal, nasal, intravenous or parenteral. For example, examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. An occlusive dressing carrier may be used to increase skin permeability and enhance absorption. Liquid dosage forms for oral administration can generally be formulated into liposome solutions containing the liquid dosage forms.

  For therapeutic or prophylactic use, pharmaceutical compositions comprising a TrkB agonist antibody may be administered locally or systemically at a therapeutically effective amount or dose. For example, the composition can be administered parenterally, enterally, injected, rapid infusion, nasopharyngeal absorption, skin absorption and orally. However, in preferred embodiments, topical administration of the composition is desired to achieve the intended therapeutic effect. In some of such embodiments, the composition can be administered to a subject in need of treatment by intravitreal injection. This can be done according to standard procedures known in the art. For example, Russelakis-Carneiro et al. , Neuropathol. Appl. Neurobiol. 25: 196-206, 1999; and Wray et al. , Arch. Neurol. 33: 183-5, 1976. Other topical administration routes that can be used to deliver the pharmaceutical composition of the present invention to the eye of a subject include, for example, intraocular, intraorbital, subconjunctival, subretinal or transscleral routes. It is envisioned that in a local mode of administration, the potential for the occurrence of side effects (eg, systemic toxicity) that can occur upon systemic administration can be reduced or eliminated. In some other embodiments, the pharmaceutical compositions of the present invention may also be systemically administered to a patient, for example by oral or parenteral routes. Parenteral routes include, for example, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes.

  A therapeutically effective amount means an amount sufficient to reduce or eliminate the symptoms of a disorder or pathological condition to be treated in a subject. In the practice of the present invention, the amount of TrkB agonist antibody administered should be effective to retard, stop or reverse ocular degeneration, eg degeneration or death of RGCs. Such an effective amount may be an ocular disorder in which the subject is affected, the stage and severity of the disorder, the general physical condition of the subject (eg, height, weight, age and health condition), the particular compound being administered. As well as from subject to subject depending on other factors. For a given TrkB agonist antibody, one skilled in the art will be able to readily identify the effective amount of the compound by using routinely practiced formulation methods. Typically, dosages used in vitro can provide useful guidance in amounts useful for in situ administration of pharmaceutical compositions to determine an effective dosage for treatment of a specific disorder in a human subject Animal models can be used to do this. In most cases, an appropriate therapeutic dose can be determined by examining the maximum tolerated dose in mammalian species by clinical trials and determining a safe dosage in normal human subjects.

  In general, except under certain circumstances where high dosages may be required, the preferred dosage of the TrkB agonist antibody is usually about 0.001 to about 1000 mg, more usually about 0.01 to about 500 mg per day It is in the range. As a general rule, the amount of TrkB agonist antibody administered is the minimum dosage that reliably ensures or suppresses the pathological condition of the subject. Also, the dosage administered and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages can be administered over relatively long intervals at relatively infrequent intervals. Some subjects may continue to receive treatment until they die. In therapeutic applications, relatively high doses at relatively short intervals are required until disease progression is reduced or halted, preferably until the subject exhibits a partial or complete remission of ocular vascular disease symptoms. obtain. The subject may then be given a preventative regime. As will be readily appreciated, the above dosage ranges are intended to support and provide general guidance to the teachings herein, and are not intended to limit the scope of the present invention. Also, further guidance for the preparation and administration of the pharmaceutical compositions of the present invention is reported in the art. See, eg, Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al. McGraw-Hill Professional (10th ed., 2001); Remington: The Science and Practice of Pharmacy, edited by Gennaro, Lippincott Williams & Wilkins (20th ed. 2003); . (Ed), Lippincott Williams & Wilkins (7th Edition, 1999).

[Example]
The following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention. Other variations of the present invention will be readily apparent to those skilled in the art and are encompassed by the appended claims.

Example 1
Development of TrkB Agonist Abs Cell lines: The specific TrkB agonist antibodies exemplified herein were selected and identified by function-based screening of a combinatorial library of scFv antibodies. Detailed procedures for performing this screening are described by Zhang et al. , Proc. Natl. Acad. Sci. USA 109: 15728-33, 2012; and Zhang et al. Chem. Biol. 20: 734-41, 2013. For simplicity, a two-step method was used. First, a reporter cell line was generated by transfecting human TrkB into CHO cells using a lentiviral vector. The NFAT promoter linked to a GFP reporter was also used to transfect the cells with a reporter cassette so that a fluorescent signal is generated when activated with the cognate ligand BDNF. Second, a library of 10 ¹¹ scFv phage was used to pan the ectodomain of human TrkB. Positives in the panning process (hits) were cloned into lentiviral vectors and clones were infected into reporter cells at a multiplicity of infection (MOI) of approximately 1 (Figure 1). In the second screening, rather than using NFAT activation as readout, a reporter cassette was used in which the CRE promoter was linked to the GFP reporter.

The hits on this selection by function were collected and multiple scFv clones were obtained by deep sequencing by 3 rounds of infection / selection. This scFv sequence was cloned into the pFUSE IgG backbone (human IgG ₁ ) to generate a scFv-Fc fusion antibody. In a preliminary test of the activity of selected Trk B antibodies, ScFv-Fc constructs were transfected into HEK 293 cells. After 2 days of culture, the supernatant of each culture clone was used to examine the effect on TrkB reporter cell line activation. The results of this test are shown in FIG. As shown in the figure, the identified TrkB scFv agonist antibodies (eg, scFv CRE-30) exhibited varying degrees of activity in activating the TrkB reporter cell line.

To further characterize the activity of the identified antibodies, selected scFv-Fc antibodies were purified using a protein G affinity column and tested in a reporter cell assay to measure potency relative to BDNF (FIG. 3) ). As shown in the figure, some Abs were full agonists compared to BDNF, while others showed some agonist action. Even within the purified and tested Abs, the potency was different from 0.3-30 nM. Ab NFAT-85 showed the highest potency _(EC 50 = 1nM), showed complete effectiveness. All TrkB agonist Abs were tested for selectivity in TrkA reporter cells. Figure 4 shows that neither BDNF nor Ab activated activated TrkA, but NGF gave a predicted robust response, confirming selectivity.

  The amino acid sequences of some of the selected TrkB agonist scFv antibodies are shown in Table 1. Antibody clones identified by screening with the "CRE" reporter are indicated as "CRE-" in the table and antibodies identified by screening with the "NFAT" reporter are indicated as "NFAT-". Sequences of heavy chain and light chain variable regions of scFv antibody and other sequence elements (eg, CDRs) are indicated by the sequence identifiers shown in Table 2.

各ｓｃＦＶ配列は、ＧＳリッチリンカー（イタリック体）によって軽鎖可変領域の配列に連結された重鎖可変領域の配列を含む。重鎖ＣＤＲの配列に下線を付し、軽鎖ＣＤＲの配列に二重下線を付している。

Table 1. TrkB agonist scFV antibody identified by functional screening

Each scFV sequence comprises the sequence of the heavy chain variable region linked to the sequence of the light chain variable region by a GS rich linker (italic). The heavy chain CDR sequences are underlined and the light chain CDR sequences are double underlined.

Example 2
Function of TrkB Agonist Antibodies in Signaling Further characterization of TrkB signaling by agonistic Abs was performed in CHO reporter cells and compared to that of BDNF. Agonist Abs, although to varying degrees, stimulated P-PLCγ, P-Akt and P-MAPK phosphorylation signaling at 5 and 30 min (Fig. B5); levels and time course were BDNF and Although varied, the signal intensity tended to correlate with the effect on the fluorescent signal in the reporter cells (see FIG. 5).

  Classical signaling of BDNF / TrkB involves a phosphorylation cascade through the Akt, MAPK and PLCγ pathways. The functions of BDNF, such as survival, differentiation, neurite outgrowth and synaptic plasticity, are attributable to various aspects of these pathways. The identified TrkB agonist Abs were also tested for trophic activity and signal transduction in primary neurons. Ab NFAT-85, NFAT-44 and NFAT-27 were found to maintain mouse sensory neuron survival similar to BDNF (not shown) in culture for 3 days. Incubation of mouse cortical neurons with Ab NFAT-85 resulted in time-dependent tyrosine phosphorylation of TrkB. However, the pattern of TrkB phospho-protein was different from that of BDNF, probably indicating the cleavage product of TrkB (Figure 6). The possibility of differential signaling by TrkB agonist Abs is currently under investigation.

  To examine downstream targets for BDNF activation, the synaptic protein Arc was measured in primary mouse cortical neurons after treatment with Ab or BDNF. Arc was initially identified as a cytoskeleton associated protein. Recent studies have linked transient increases of Arc expression in dendrite spines to stable expansion of the F-actin network, which is thought to underlie synaptic morphological expansion and stable LTP. ing. BDNF, which has long been involved in synaptic plasticity and memory retention, activates Arc-dependent immobilization and is required for actin-dependent spine elongation. Current evidence further suggests the possibility of reciprocal interactions of F-actin formation, transformation upon Arc and LTP immobilization. The results of this test are shown in FIG. Similar to BDNF, antibody NFAT-85 stimulated Arc after 2 to 4 hours of incubation.

  Since anti-TrkB agonist Abs were found by panning and selection with human TrkB receptors, we examined the relative potency of Abs on rodent cells for future in vitro and in vivo studies. To examine this, we used cells of a mouse embryonic stem cell line previously shown to respond to BDNF and compared TrkB phosphorylation stimulated by Ab NFAT-85 and BDNF. Ab NFAT-85 was found to increase phosphorylation in a concentration dependent manner. However, the potency was at least two orders of magnitude smaller than the effect on human reporter cells (about 1 nM). In both mouse and human cells, BDNF resulted in robust signaling at approximately 2 nM (FIG. 8).

  Taken together, testing of signal transduction and downstream markers is consistent with TrkB binding in a selective manner by agonist Abs. Concentration-response in mouse cells defines the relative potency of Ab NFAT-85 for further rodent testing.

[Example 3]
Effect of TrkB Agonist Ab in Retinal Culture System In order to start a study on the effect of TrkB agonist on retinal physiology in vitro, a mouse retinal explant culture system was established. Briefly, the retinas of adult mice were dissected out and placed in culture medium (with RGC layer facing up) for a period of 0-14 days. By definition, RGCs are axotomized; during this time there is a progressive decrease of the various retinal layers and RGC dendritic fields. Retraction of RGC dendrites becomes apparent as early as 6 hours in culture as assessed by shawl analysis, which measures dendrite area parameters as a function of distance from the cell body. By day 3 in culture, there is a significant reduction in dendrite crossing and dendrite branching.

  The 3-day culture period provides a good dynamic range to test for the neuroprotective or neuroregenerative effects of BDNF and TrkB agonist Abs on the dendritic tree of RGCs. Incubation of explants with 100 ng / ml BDNF significantly blocks dendrite retraction, resulting in explants with zero area under the shawl and dendritic branches after 3 days in culture. It was observed to be similar to the ones, suggesting complete protection of the dendrite's regression response to axotomy. To determine whether BDNF could reverse axotomy-induced dendrite retraction, explants were maintained in culture for 3 days at which time BDNF was added and shawl analysis performed. Notably, the delayed application of BDNF reverses the regression response to the parameters, again similar to that of explants with zero time-point, and BDNF induces the sprouting response of neural regeneration in injured neurons Was suggested.

  This paradigm establishes the basis for testing whether TrkB agonist Abs can suppress dendrite regression in retinal explants similar to those observed with BDNF. FIG. 9 shows that Ab NFAT-85 maintained the dendrite parameters of axotomized RGC dendrites with the same size as BDNF and was observed with Ab NFAT-85 in other cell types Biochemical and signaling parameters were validated. Future experiments will examine the effects of delayed administration of Ab NFAT-85, as well as the persistence of the effects of agonist Abs in this culture system.

  In confirmation of the above unpublished results, two recent reports have shown the nutritional effects of BDNF on RGCs. First, Johnson et al. According to (Invest. Ophthalmol. Vis. Sci. 57: 253-264, 2016), when BDNF + CNTF (ciliary neurotrophic factor) is administered to mouse retinal explants, the branched segments, junctions and RGCs on day 7 It has been shown that the number of terminal branches can be improved. Also, Domenici et al. According to (PloS One 9 (12): e115579, 2014), intravitreal application or transsurface application of BDNF produces visual function during electrophysiological evaluation (not shown) in the DBA / 2J mouse model of spontaneous glaucoma It was shown to be improved. Interestingly, these effects occurred independently of the rise in IOP.

Example 4
Efficacy of TrkB Agonist Ab in a Rat Model of Glaucoma.
Glaucoma ocular hypertensive (OHT) modeling was performed in Brown Norway rats by injecting sterile 5 μm magnetic microbeads into the anterior chamber. The experimental procedure is essentially as described by Samsel et al. , Invest. Ophthalmol. 52: 1671, 2011; and Morgan and Tribble, Exp. Eye Res. 141: 9-14, 2015 as described. A magnet was used to attract the beads to the iridocorneal canal to break the trabecular meshwork, inhibit vitreous outflow, and subsequently raise the intraocular pressure (IOP). The rise in IOP was measured periodically with a rebound tonometer. Bead injections were performed on the left eye and the right eye was used as a control.

  Five weeks after bead injection, rat left eye was administered by intravitreal injection phosphate buffered saline (PBS), 500 ng of Trk B agonist antibody NFAT-85 (Ab 85) or 500 ng of BDNF in a volume of 1 μl. In some animals, the effects of Ab85 or BDNF were examined in normal eye (NT) eyes (not receiving magnetic microbeads injection). Two weeks later (7 weeks after microbead injection), rats were sacrificed, the retinas removed and prepared for Diolistic labeling of retinal ganglion cells and their dendrites. Tree morphology (dendrite bifurcation index, dendrite length and dendrite crossover) was quantified by shawl analysis. An average of 6-10 cells were analyzed per retina. An overview of the experimental paradigm is shown in FIG.

  FIG. 11 shows the average IOP and peak IOP levels (in mmHg) of rats in the OHT and NT groups treated with PBS, Ab85 or BDNF. In this method, IOP was elevated early after microbead injection, remained high for about 3 weeks and returned to normal levels by 4 weeks.

  FIG. 12 shows quantification of the average of dendrite tree crossings as assessed by shawl analysis. The top graph is a comparison of OHT rats treated with various agents compared to controls that are eyes with normal intraocular pressure (NT). Both BDNF and Ab 85 (which are more pronounced) show increased cross over compared to PBS treated eyes and not different from NT eyes. The lower plot shows that treatment of NT eyes with BDNF or Ab 85 did not significantly alter the mean crossover (plotted with OHT as a comparison point).

  FIG. 13 shows a statistical comparison of the area under the curve (AUC), branching index and dendrite length in the shawl analysis shown in FIG. All measurements were significantly lower in OHT + PBS rats compared to NT eyes. Also, with either OHT + Ab85 or BDNF, these measurements were ameliorated to an equivalent value of NT. Ab 85 treatment and NT eyes without BDNF treatment were different from NT control.

  The results of this study show the positive effect of the regenerative nature of the TrkB agonist Ab on the dendrites of the retinal ganglion neurons of the rat in the hypertensive glaucoma model. Importantly, this regenerative effect occurs after induction of the injury caused by elevated intraocular pressure, similar to the post-lesion treatment paradigm that has been clinically practiced for glaucoma.

[Example 5]
Bispecific Antibodies Targeting TrkB and TNF We next make bispecific antibodies from the TrkB agonist antibody Ab85 described herein and the well known anti-TNF antibody Humira (adalimumab) Attempts were made to generate a proof-of-concept bispecific antibody construct. This bispecific molecule construct contains one arm of Ab85 (TrkB antibody) and the other arm of adalimumab. Briefly, the Ab85 scFv fragment was fused to a human IgG1 Fc fragment carrying the Y407T mutation. The obtained fusion protein is shown in SEQ ID NO: 74 (Ab85-Fc (407)). Meanwhile, the adalimumab Fab heavy chain was fused with a human IgG1 Fc fragment with a T366Y mutation. The resulting fusion protein is shown in SEQ ID NO: 75 (adalimumab-heavy chain-Fc (366)).

  These two fusion sequences (Ab85-Fc (407), adalimumab-heavy chain-Fc (366)), together with the light chain coding encoding vector for adalimumab, for bispecific antibody expression and synthesis. Transfect together into Expi 293 cells. The synthesized antibodies can then be purified by protein G affinity chromatography.

SEQ ID NO: 74 (Ab85-Fc (407)
MAQVQLQQSGPGLVKPSQTLSLTCVISGDSVSDNSGAWNWIRQSPSRGLEWLGRTYYRSKWYTDYADSVKSRITIIPDIPKNQFSLHLNSVTPEDTAVYYCVRGYYYAFHIWGQGTMVTVSSGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRTYYASWYQQKPGQAPVLVAAGKNNRPSGIPDRFSASSSGNTASLTITGAQAEDEADYYCSSRDSSRSHHLLFGGGTKVTVLGGLGGLASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 75 (adalimumab-heavy chain-Fc (366))
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be appreciated by those skilled in the art in light of the teachings of the present invention that it departs from the spirit and scope of the appended claims. It will be readily apparent that without departing from the scope of the present invention several changes and modifications can be made.

  All publications, databases, GenBank sequences, patents and patent applications cited herein are hereby incorporated by reference as if each were specifically and individually indicated.

Claims (36)

  An antibody or antigen-binding fragment that specifically binds to tropomyosin receptor kinase B (Trk B), having the same binding specificity as that of the second antibody, said second antibody each comprising (1) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and SSRDSSRSHHLL (SEQ ID NO: 73); (2) GYSFTSYW (SEQ ID NO: 46) ), IYPGDSDT (SEQ ID NO: 47), ARQGASSTSYDY (SEQ ID NO: 48), QSISSY (SEQ ID NO: 49), AAS and QQANSFPVA (SEQ ID NO: 50); (3) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 51) Number: 52), VRQMLF (sequence number : 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAWV (SEQ ID NO: 56); (4) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 52) Number: 53), SGINVGAYR (SEQ ID NO: 57), YKTDSDK (SEQ ID NO: 58) and AIWH SSAWV (SEQ ID NO: 56); (5) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 52) SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 55), AIWH SSAWV (SEQ ID NO: 56); (6) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (Array No. 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHS SACV (SEQ ID NO: 59); (7) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), ARQLLY (SEQ ID NO: 52) SEQ ID NO: 60), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 61), AIWH SSAWV (SEQ ID NO: 56); (8) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ATRVLPAGHF (SEQ ID NO: 62), NIGDKF (SEQ ID NO: 63), YDS and QVWDNSSNQGV (SEQ ID NO: 64); (9) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67) ) SLRTYY (SEQ ID NO: 68), GKN and NSRDGSGNNV (SEQ ID NO: 69); or (10) GYTFTSYA (SEQ ID NO: 70), INTNTGNP (SEQ ID NO: 71), ASRAAAGFDY (SEQ ID NO: 72), SGINVGTYR (Sequence No. 54) An antibody or antigen binding fragment comprising the sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 identical to YKSDSDK (SEQ ID NO: 55) and AIWHSSAWV (SEQ ID NO: 56).   The heavy chain variable region sequence and the light chain variable region sequence, one or both of which (1) SEQ ID NO: 38 and SEQ ID NO: 39; (2) SEQ ID NO: 14 and SEQ ID NO: 15; 3) SEQ ID NO: 16 and SEQ ID NO: 17; (4) SEQ ID NO: 18 and SEQ ID NO: 19; (5) SEQ ID NO: 20 and SEQ ID NO: 21; (6) SEQ ID NO: 22 and SEQ ID NO: 23 (7) SEQ ID NO: 24 and SEQ ID NO: 25; (8) SEQ ID NO: 26 and SEQ ID NO: 27; (9) SEQ ID NO: 28 and SEQ ID NO: 29; (10) SEQ ID NO: 30 and SEQ ID NO: (11) SEQ ID NO: 32 and SEQ ID NO: 33; (12) SEQ ID NO: 34 and SEQ ID NO: 35; or (13) Sequence of heavy chain variable region shown in SEQ ID NO: 36 and SEQ ID NO: 37 And the light chain variable region are identical. Antigen-binding fragment.   (1) SEQ ID NO: 38 and SEQ ID NO: 39; (2) SEQ ID NO: 14 and SEQ ID NO: 15; (3) SEQ ID NO: 16 and SEQ ID NO: 17; (4) SEQ ID NO: 18 and SEQ ID NO: (5) SEQ ID NO: 20 and SEQ ID NO: 21; (6) SEQ ID NO: 22 and SEQ ID NO: 23; (7) SEQ ID NO: 24 and SEQ ID NO: 25; (8) SEQ ID NO: 26 SEQ ID NO: 27; (9) SEQ ID NO: 28 and SEQ ID NO: 29; (10) SEQ ID NO: 30 and SEQ ID NO: 31; (11) SEQ ID NO: 32 and SEQ ID NO: 33; The antibody or antigen-binding fragment according to claim 1, comprising the heavy chain variable region sequences and the light chain variable region sequences shown in SEQ ID NO: 34 and SEQ ID NO: 35; or (13) SEQ ID NO: 36 and SEQ ID NO: 37.   (1) VSDNSGAWN (SEQ ID NO: 65), YRSKWYT (SEQ ID NO: 66), VRGY YYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and SSRDSSRSHHLL (SEQ ID NO: 73); (2) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ARQGASS TSY DY (SEQ ID NO: 48), QSISSY (SEQ ID NO: 49), AAS and QQANSFPVA (SEQ ID NO: 50); (3) GFIFSRYN (SEQ ID NO: 51) ), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAV (SEQ ID NO: 56); (4) GFIFSRYN (SEQ ID NO: 56) Column number: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKTDSDK (SEQ ID NO: 58) and AIWHSSAV (SEQ ID NO: 56); (5) GFIFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 55), AIWHSSAV (SEQ ID NO: 56); GIFFSRYN (SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), VRQMLF (SEQ ID NO: 53), SGINVGTYR (SEQ ID NO: 54), YKSSDDK (SEQ ID NO: 55) and AIWHSSACV (SEQ ID NO: 59); ) GFIFSRYN SEQ ID NO: 51), INTDGSVI (SEQ ID NO: 52), ARQLLY (SEQ ID NO: 60), SGINVGAYR (SEQ ID NO: 57), YKSDSDK (SEQ ID NO: 61), AIWHSSAWV (SEQ ID NO: 56); (8) GYSFTSYW (SEQ ID NO: 46), IYPGDSDT (SEQ ID NO: 47), ATRVLPAGHF (SEQ ID NO: 62), NIGDKF (SEQ ID NO: 63), YDS and QVWDNSSNQGV (SEQ ID NO: 64); (9) VSDNSGAWN (SEQ ID NO: 65) ), YRSKWYT (SEQ ID NO: 66), VRGYYYAFHI (SEQ ID NO: 67), SLRTYY (SEQ ID NO: 68), GKN and NSRDGSGNNVV (SEQ ID NO: 69); or (10) GYTFTSYA (SEQ ID NO: 70), INTNTG A heavy chain CDR1 substantially identical to NP (SEQ ID NO: 71), ASRRAAAGFDY (SEQ ID NO: 72), SGINVGTYR (SEQ ID NO: 54), YKSDSDK (SEQ ID NO: 55) and AIWHSSAW (SEQ ID NO: 56) The antibody or antigen-binding fragment according to claim 1, comprising the sequences of 3 and light chain CDRs 1-3.   The antibody or antigen-binding fragment according to claim 1, comprising a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 46-48, 51-53, 60, 62, 65-67 and 70-72.   The antibody or antigen-binding fragment according to claim 5, further comprising a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 49, 50, 54-59, 61, 63, 64, 68, 69 and 73.   (1) SEQ ID NO: 70 to 72 (2) SEQ ID NO: 46 to 48 (3) SEQ ID NO: 51 to 53 (4) SEQ ID NO: 51, 52, 60 (5) SEQ ID NO: 46 , 47 and 62, or (6) heavy chain CDR1 to 3 sequences identical to SEQ ID NO: 65 to 67.   (1) SEQ ID NO: 65 to 68, GKN and SEQ ID NO: 73; (2) SEQ ID NO: 46 to 49, AAS and SEQ ID NO: 50; (3) SEQ ID NO: 51 to 56; (4) SEQ ID NO: (5) SEQ ID NOs: 51-53, 57, 55 and 56; (6) SEQ ID NOs: 51-55 and 59; (7) SEQ ID NOs: 51, 52, 60: 51-53, 57, 58 and 56; 57, 61 and 56; (8) SEQ ID NO: 46, 47, 62, 63, YDS and SEQ ID NO: 64; (9) SEQ ID NO: 65 to 68, GKN and SEQ ID NO: 69; or (10) Sequence The antibody or antigen-binding fragment according to claim 7, comprising the sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 shown in Nos. 70 to 72 and 54 to 56.   2. A sequence of a light chain CDR selected from the group consisting of SEQ ID NOs: 49, 50, 54-59, 61, 63, 64, 68, 69 and 73 and the sequences AAS, YDS and GKN. Antibody or antigen binding fragment.   The antibody or antigen-binding fragment according to claim 9, further comprising a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 46-48, 51-53, 60, 62, 65-67 and 70-72.   (1) SEQ ID NO: 68, GKN and SEQ ID NO: 73; (2) SEQ ID NO: 49, AAS and SEQ ID NO: 50; (3) SEQ ID NO: 54 to 56; (4) SEQ ID NO: 57, 58 And 56; (5) SEQ ID NO: 57, 55 and 56; (6) SEQ ID NO: 54, 55 and 59; (7) SEQ ID NO: 57, 61 and 56; (8) SEQ ID NO: 63, YDS and Sequence 10. The antibody or antigen binding fragment according to claim 9, comprising sequences of light chain CDRs 1 to 3 which are identical to SEQ ID NO: 68, GKN and SEQ ID NO: 69;   The antibody according to claim 1, comprising a heavy chain variable region substantially identical to SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38. Antigen binding fragment.   The antibody or antigen-binding fragment according to claim 1, which is a scFv fragment, wherein the heavy chain and light chain variable region sequences are linked by a linker sequence.   14. The antibody or antigen binding fragment of claim 13, wherein the linker sequence comprises the sequence set forth in any one of SEQ ID NOs: 40-45.   The antibody or antigen-binding fragment according to claim 13, comprising the sequence shown in any one of SEQ ID NOs: 1-13.   IgAl, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, F (ab) 2, Fv, scFv, IgGACH2, F (ab ') 2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2 The antibody or antigen-binding fragment according to claim 1, which is, dsFv, Fv, scFv-Fc, (scFv) 2, non-depleting IgG, diabody and bivalent antibody.   The antibody or antigen-binding fragment according to claim 16, which is an IgG selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and synthetic IgG.   The antibody or antigen-binding fragment according to claim 16, which is Fab, scFv or dsFv.   17. The antibody or antigen binding fragment of claim 16 conjugated to a synthetic molecule.   A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen binding fragment according to claim 1 and a pharmaceutically acceptable carrier.   A bispecific compound comprising the antibody or antigen binding fragment of claim 1.   A kit comprising the antibody or antigen-binding fragment of claim 1.   A polynucleotide encoding the heavy chain and / or light chain variable region of the antibody or antigen-binding fragment according to claim 1.   A vector comprising the polynucleotide of claim 23.   A method for promoting survival, synaptic function or regeneration of retinal ganglion cells, motor neurons or neurons of the central nervous system (CNS) of a subject, wherein a therapeutically effective amount of the subject according to claim 1 is used. 26. A method comprising administering a pharmaceutical composition comprising a TrkB agonist antibody or antigen binding fragment or the vector according to claim 24, thereby promoting survival or regeneration of retinal ganglion cells of said subject.   26. The method of claim 25, wherein the pharmaceutical composition is administered to one or both eyes of a subject.   26. The method of claim 25, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.   26. The method of claim 25, wherein the subject suffers from or is at risk of developing a degenerative disorder of the eye, a motor neuron disease or a degenerative disorder of the central nervous system (CNS).   The above eye degeneration disorders include glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion, central retinal vein occlusion, diabetic neuropathy, age-related macular degeneration (AMD), anterior ischemic optic neuropathy (AION) The method according to claim 28, wherein the retinopathy is diabetic retinopathy.   The motor neuron diseases include ALS, idiopathic motor neuropathy, hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP) and pseudospheres 29. The method of claim 28, which is paralysis, Bell's palsy or spinal muscular atrophy (SMA).   29. The method of claim 28, wherein the degenerative disease of the central nervous system (CNS) is Alzheimer's disease, Parkinson's disease or Huntington's disease.   25. A method for treating or preventing a degenerative pathological condition of a subject's eye, said antibody or antigen-binding fragment according to claim 1 or a therapeutic effective amount according to claim 24 for said subject. Administering a pharmaceutical composition comprising a vector, thereby treating or preventing optic neuropathy in said subject.   33. The method of claim 32, wherein the pharmaceutical composition is administered to one or both eyes of the subject.   33. The method of claim 32, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.   33. The method of claim 32, wherein the subject is affected by or at risk of developing a degenerative condition of the eye.   Said degenerative pathological condition is glaucoma, optic nerve injury, optic neuritis, optic neuropathy, central retinal artery occlusion, central retinal vein occlusion, diabetic neuropathy, age-related macular degeneration (AMD), anterior ischemic 36. The method of claim 35, wherein the optic neuropathy (AION) or diabetic retinopathy.

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